Megakaryocytes are the hematopoietic cells, largely found in the bone marrow, but also in peripheral blood and perhaps other tissues as well, which produce platelets (also known as thrombocytes) and subsequently release them into circulation. Megakaryocytes, like all of the hematopoietic cells of the human hematopoietic system, ultimately derive from a primitive pluripotent marrow stem cell after passing through a complex pathway comprising many cellular divisions and considerable differentiation and maturation. Mature megakaryocytes ultimately undergo subdivisions and release the cytoplasmic fragments which are circulating platelets.
The platelets derived from these megakaryocytic cells are critical for initiating blood clot formation at the site of injury Platelets also release growth factors at the site of clot formation that speed the process of wound healing and may serve other functions. Clinical experience has shown that control mechanisms exist to maintain effective platelet numbers in humans, but that at times these specific controls are either inadequate or ineffective and lead to depressed levels of platelets (thrombocytopenia) or thrombocytosis despite normal numbers of red blood cells and white blood cells.
The inability to form clots is the most immediate and serious consequence of a low platelet count, a potentially fatal complication of many therapies for cancer. Such cancer patients are generally treated for this problem with platelet transfusions. Other patients frequently requiring platelet transfusions are those undergoing bone marrow transplantation or patients with aplastic anemia.
Platelets for such procedures are obtained by plateletphoresis from normal donors Like most human blood products, platelets for transfusion have a relatively short shelf-life and also expose the patients to considerable risk of exposure to dangerous viruses, such as the human immunodeficiency virus (HIV) or the various hepatitis viruses.
The ability to stimulate endogenous platelet formation in thrombocytopenic patients with a concomitant reduction in their dependence on platelet transfusion would be of great benefit. In addition the ability to correct or prevent thrombocytopenia in patients undergoing radiation therapy or chemotherapy for cancer would make such treatments safer and possibly permit increases in the intensity of the therapy thereby yielding greater anti-cancer effects.
For these reasons considerable research has been devoted to the identification and purification of factors involved in the regulation of megakaryocyte and platelet production. Although there is considerable controversy, the factors regulating the growth and differentiation of hematopoietic cells into mature megakaryocyte cells and the subsequent production of platelets by these cells are believed to fall into two classes.
Megakaryocyte colony-stimulating factors (meg-CSFs) are the first group of regulatory factors which function to support the proliferation and differentiation of megakaryocytic progenitors (CFU-M) in culture. The second group of factors have been defined by their activity towards megakaryocytes in either in vivo or in vitro bioassays. Factors which elicit an in vivo response, such as an increase in the circulating level of platelets have been defined as thrombopoietin ("TPO"). Factors which support either the differentiation, maturation or development of megakaryocytes in an in vitro culture system have been termed megakaryocyte stimulating activity, megakaryocyte potentiating activity, or thrombopoietin-like activity. It is unclear whether thrombopoietic factors are structurally identical or related to any of the in vitro defined megakaryocyte stimulating activities.
From the studies reported to date, it is not clear whether activities identified as meg-CSF also have TPO activity or vice versa. Many different reports in the literature describe factors which interact with cells of the megakaryocytic lineage and report megakaryocyte growth promoting activities specific for the megakaryocyte lineage. [See, e.g., E. Mazur, Exp. Hematol., 15:340-350 (1987); N. Williams et al, J. Cell. Physiol., 110:101-104 (1982); J. E. Straneva et al, Exp. Hematol., 14:919-929 (1986)]. An understanding of the specifics of positive and negative control of megakaryocytopoiesis is incomplete.
For example, human IL-3 supports human megakaryocyte colony formation and, at least in monkeys, also frequently elicits an elevation in platelet count. However, IL-3 influences hematopoietic cell development in all of the hematopoietic lineages and can be distinguished from specific regulators of megakaryocytopoiesis and platelet formation which interact selectively with cells of the megakaryocytic lineage.
There is strong evidence that in mice, murine IL-6 has thrombopoietin activity in vivo and augments murine megakaryocyte colony formation with IL-3 in in vitro bioassays. However the thrombopoietic effect is not striking (50-60% increase in circulating platelet numbers in 5 days) [T. Ishibashi et al, J. Clin. Invest., 79:286-289 (1987); T. Ishibashi et al, Blood, 74(4):1241-1244 (1989); T. Ishibashi et al, Proc. Natl. Acad. Sci. USA, 86:5953-5957 (1989)]. In vivo administration of IL-6 to mice also increases megakaryocyte size and ploidy. There is much less evidence that IL-6 has TPO-like or megakaryocyte potentiating activity in human in vitro assays [see, e.g., E. Bruno and R. Hoffman, Exp. Hematol., 17:1038-4 (1989)]. In most of these assays human IL-6 has shown no TPO-like activity [M. Teramura et al, Exp. Hematol., 17:1011-1016 (1989) and M. W. Long et al, J. Clin. Invest., 82:1779-1786 (1988)].
R. Hoffman et al, J. Clin. Invest., 75:1174-1182 (1985) describes using a human megakaryocyte colony assay to purify from serum a colony stimulating activity with an apparent MW of 46,000. This factor is found in the 70-80% ammonium sulfate cut, binds to wheat germ lectin, and loses activity after deglycosylation. A similar activity was detected in thrombocytopenic rabbit plasma that increases the incorporation of .sup.75 Se methionine into platelets in mice. This activity was purified 7,000 fold from plasma, but contaminating proteins were present as determined by SDS-PAGE electrophoresis. See, e.g., R. Hill and J. Levin, Exp. Hematol., 14:752-759 (1986). Other serum derived factors are described by J. E. Straneva et al, Exp. Hematol., 15:657-663 (1987); and E. Mazur et al, Exp. Hematol., 13:1164-1172 (1985].
Megakaryocyte growth promoting activities, and thrombopoietin also have been derived from human embryonic kidney (HEK) cells [See, e.g., T. P. McDonald, Exp. Hematol., 16:201-205 (1988); T. P. McDonald et al, Biochem. Med. Metab. Biol., 37:335-343 (1987); G. Tayrien and R. D. Rosenberg, J. Biol. Chem., 262:3262-3268 (1987) and others]. Each has purified to homogeneity a 15,000 molecular weight activity that readily dimerizes to 30,000 molecular weight HEK-derived activity can increase isotopic incorporation into platelets when given parenterally in mice, and increase the production of platelet factor 4-like proteins in rodent megakaryocyte lineage cells. This activity is heat stable, and maintains activity after treatment with endoglycosidases, and binds to wheat germ lectin.
Finally, activities have been described from urine that promote megakaryocyte growth in rodents in vivo and in marrow culture. Kawakita has partially purified an activity from urine that varies with patient platelet count, and under dissociating conditions has a molecular weight of 45,000. The activity of this on human megakaryocyte progenitors has not been tested, nor has it been shown to be specific for the megakaryocyte hematopoietic lineage. [M. Kawakita et al, Br. J. Haem., 62:715-722 (1986); M. Kawakita et al, Blood, 61:556-560 (1983); see, also, S. Kuriya et al, Exp. Cell Biol., 55:257-264 (1987); K. Enomoto et al, Brit. J. Haem., 45:551-556 (1980)].
Despite such reports tentatively identifying such regulatory factors, the biochemical and biological identification and characterization of these factors has been hampered by the small quantities of the naturally occurring factors available from natural sources, e.g., blood and urine.
There remains a need in the art for the isolation, identification and production of additional proteins purified from their natural sources or otherwise produced in homogeneous form, which are capable of stimulating or enhancing the production of platelets in vivo, to replace presently employed platelet transfusions and otherwise useful in the treatment and/or diagnosis of blood and blood platelet disorders.